Journal: PLoS ONE

Article Title: TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

doi: 10.1371/journal.pone.0082539

Figure Lengend Snippet: ( A ) Scheme of the architecture of TALE–PvuII fusion proteins. Left: wtPvuII, a homodimer in which the DNA-binding module of a TALE protein is fused via a linker of defined length. Right: scPvuII, a monomeric nuclease in which the DNA-binding module of a TALE protein is fused via a linker of defined length. ( B ) Model of a TALE–wtPvuII fusion protein. The fusion protein is a dimer of identical subunits, each composed of a PvuII subunit and a TALE protein. This model was constructed by aligning the structures of the individual proteins [pdb 1pvi and pdb 3ugm ] on a DNA composed of the PvuII recognition site and two TALE target sites up- and downstream of the PvuII recognition site, separated by 6 bp. The C-termini of the PvuII subunits and the N-termini of the TALE protein are separated by about 3 nm. This distance must be covered by a peptide linker of suitable length. The image was generated with PyMol.

Article Snippet: PCR amplicons were cleaned up using the QIAquick PCR Purification Kit (Qiagen) and 100 ng DNA was subjected to digestion with 20 U of PvuII-HF (New England BioLabs).

Techniques: Binding Assay, Construct, Generated

Journal: PLoS ONE

Article Title: TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

doi: 10.1371/journal.pone.0082539

Figure Lengend Snippet: ( A ) and ( B ) Comparison of the cleavage rates of selected AvrBs3-PvuII fusion proteins (as indicated) under low ionic strength: 76 mM (20 mM Tris-Ac, 50 mM K-Ac, 2 mM Mg-Ac, pH 7.5). In the top row the cleavage of the addressed substrate (T3-6bp-P-6bp-T3) is shown, in the bottom row that of the unaddressed substrate (-P-). All cleavage experiments were done with 8 nM DNA and 8 nM enzyme. ( C ) Comparison of the cleavage rates of an unaddressed substrate by selected AvrBs3-PvuII fusion proteins (as indicated) under physiological ionic strength: 143 mM (20 mM Tris-Ac, 120 mM K-Ac, 1 mM Mg-Ac, pH 7.5). The experiments were done with an excess of enzyme, the TALE-scPvuII fusion protein (top, 60 nM enzyme, 6 nM DNA) shows a higher cleavage activity with an unaddressed substrate (-P-) than the homodimeric TALE-PvuII T46G fusion protein (bottom, 80 nM enzyme, 8 nM DNA). See the appearance of nicked and linearized DNA with AvrBs3-28-L-scPvuII T46G . There is no nicking or cleavage detectable of the unaddressed substrate with AvrBs3-28-L-PvuII T46G . oc, open circle; lin, linearized; sc, supercoiled.

Article Snippet: PCR amplicons were cleaned up using the QIAquick PCR Purification Kit (Qiagen) and 100 ng DNA was subjected to digestion with 20 U of PvuII-HF (New England BioLabs).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

doi: 10.1371/journal.pone.0082539

Figure Lengend Snippet: ( A ) Competition cleavage experiments with AvrBs3-28-L-PvuII T46G under physiological ionic strength. Shown is the cleavage pattern with supercoiled plasmid DNA with an addressed site (8 nM) in competition with a PCR fragment (unP) with an unaddressed site (32 nM). The experiment was carried out with a variable excess of enzyme over plasmid substrate (0.25 to 40-fold). The enzyme shows complete cleavage of the addressed substrate but no cleavage of the unaddressed substrate, even in an overnight incubation with a 40-fold excess of enzyme over the addressed plasmid substrate (8 nM) and 10-fold excess over the unaddressed PCR substrate (32 nM). The brackets indicate the positions where one would expect the products of cleavage of the unaddressed PCR substrate. oc, open circle; lin, linearized; sc, supercoiled. ( B ) Quantitative determination of the preference of AvrBs3-28-L-PvuII T46G for an addressed (T3-6bp-P-6bp-T3) over an unaddressed site (-P-). The reactions were performed in triplicate under physiological conditions with 20 nM enzyme and 20 nM addressed substrate (squares) and unaddressed substrate (circles), both PCR fragments were radioactively labelled with [α 32 P]dATP. The insert shows the primary data: the electrophoretic analysis of the cleavage reaction products using an Instant Imager. From the fit, a cleavage preference of > 34,000-fold was determined.

Article Snippet: PCR amplicons were cleaned up using the QIAquick PCR Purification Kit (Qiagen) and 100 ng DNA was subjected to digestion with 20 U of PvuII-HF (New England BioLabs).

Techniques: Plasmid Preparation, Incubation

Journal: PLoS ONE

Article Title: TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

doi: 10.1371/journal.pone.0082539

Figure Lengend Snippet: ( A ) Specificity of cleavage analyzed with the T3-6bp-P-6bp-T3 substrate and the T4-6bp-P-6bp-T4 substrate which differ in 11 (8, respectively, considering the degeneracy of the TALE recognition code) out of 19 positions from the AvrBs3 target site. No nicking or cleavage of the AvrBs4 substrate (8 nM) by AvrBs3-28-L-PvuII T46G (8 nM) could be detected. ( B ) Cleavage of a “half-site” substrate by AvrBs3-28-L-PvuII T46G . The “half-site” substrate is a bipartite substrate consisting of an AvrBs3 recognition site and a PvuII recognition site (T3-6bp-P). The sc plasmid (8 nM) with the “half-site” was incubated with an equimolar concentration of AvrBs3-28-L-PvuII T46G (8 nM). The assay was done under physiological ionic strength and in competition with a 32 nM PCR fragment (unP) with one unaddressed PvuII site (-P-). Whereas the “half-site” substrate is cleaved almost to completion, the unaddressed PCR fragment is not cleaved at all. ( C ) The effect of the distance of the AvrBs3 and the PvuII site on the rate of DNA cleavage by various AvrBs3-PvuII fusion proteins. 20 nM radioactively labelled PCR fragments with 2 (T3-2-P-2-T3), 4 (T3-4-P-4-T3), 6 (T3-6-P-6-T3) and 8 (T3-8-P-8-T3) bp between the AvrBs3 and the PvuII site were incubated with 20 nM AvrBs3-28-L-PvuII T46G , AvrBs3-28-PvuII T46G and AvrBs3-L-PvuII T46G for 60 min.

Article Snippet: PCR amplicons were cleaned up using the QIAquick PCR Purification Kit (Qiagen) and 100 ng DNA was subjected to digestion with 20 U of PvuII-HF (New England BioLabs).

Techniques: Plasmid Preparation, Incubation, Concentration Assay

Journal: PLoS ONE

Article Title: TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

doi: 10.1371/journal.pone.0082539

Figure Lengend Snippet: ( A ) PCR was performed with the plasmid from the HEK293 cells resulting in a DNA fragment of 517 bp. * indicates the cleavage site of PvuII. ( B ) Analysis of the PCR product (14.5 nM) after digestion with 20 U of PvuII for 1 h. A cleavage-resistant band indicates the loss of the PvuII site by NHEJ and confirms the activity of the TALE-PvuII fusion proteins. ( C ) Cell toxicity of the PvuII-based TALENs. After co-transfection of a mCherry expression plasmid, cell survival rate was calculated as the decrease in the number of mCherry-positive cells from day 2 to day 5 by flow cytometry, normalized to cells transfected with an I-SceI expression vector. * Statistically significant differences in toxicities between I-SceI and TALE-PvuII fusion proteins are indicated (P-values) .

Article Snippet: PCR amplicons were cleaned up using the QIAquick PCR Purification Kit (Qiagen) and 100 ng DNA was subjected to digestion with 20 U of PvuII-HF (New England BioLabs).

Techniques: Plasmid Preparation, Activity Assay, TALENs, Cotransfection, Expressing, Flow Cytometry, Transfection

Journal: PLoS ONE

Article Title: TALE-PvuII Fusion Proteins – Novel Tools for Gene Targeting

doi: 10.1371/journal.pone.0082539

Figure Lengend Snippet: With the exception of engineered homing endonucleases (“meganucleases”) in which the function of DNA binding and DNA cleavage is present in the same polypeptide chain , the other engineered nucleases consist of separate DNA-binding (green) and DNA-cleavage (blue) modules. Zinc finger nucleases and TALE nucleases usually have the non-specific cleavage domain of the restriction endonuclease FokI as DNA-cleavage module, but as shown recently and in the present paper the restriction endonuclease PvuII can also be used for this purpose . PvuII has also been employed in TFO-linked nucleases and in protein fusions (with catalytically inactive I-SceI) as DNA-cleavage module. Zinc finger nucleases, TALE nucleases and TFO-linked nucleases are programmable, as are the RNA-mediated nucleases [modified after ] .

Article Snippet: PCR amplicons were cleaned up using the QIAquick PCR Purification Kit (Qiagen) and 100 ng DNA was subjected to digestion with 20 U of PvuII-HF (New England BioLabs).

Techniques: Binding Assay, Zinc-Fingers, Modification

Journal: Nucleic Acids Research

Article Title: A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy

doi: 10.1093/nar/gkab770

Figure Lengend Snippet: High density in vivo psoralen DNA inter-strand cross-linking coupled to denaturing spreading allows to visualize the in vivo three-strand DNA structure of the mammalian mitochondrial D-loop. ( A ) Representative EM picture of a chromosomal DNA fiber subjected to denaturing spreading with a schematic representation of the regions of the fiber heavily cross-linked by psoralen in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. Enlarged views of regions of the chromosomal DNA fiber with regularly spatially closed interspaced cross-links (indicative of assembly in a tight nucleosomal array) or with more interspersed cross-links are shown (see the text). Scale bars of 360 nm (1 kbp) or 180 nm (0.5 kbp) (in black) are shown on each EM picture. ( B , C ) Representative EM pictures (and enlarged views) of circular (B) or PvuII-linearized (C) mitochondrial genomes subjected to denaturing spreading (see the text). Scale bars of 360 and 180 nm corresponding to 1 kbp and 0.5 kbp, respectively, are reported on each picture. Schematized representations are shown with heavily cross-linked regions in black and ssDNA bubbles due to the absence of DNA inter-strand cross-links in red. The position of the D-loop structure is indicated by the asterisk. ( D ) Enlarged views (EM pictures) and schematized representations of the D-loop region on PvuII-linearized mitochondrial DNA molecules subjected to denaturing spreading (see the text). Heavily cross-linked stretches of dsDNA (and ssDNA denaturation bubbles) are clearly visible in the D-loop structures opposite to the side of the D-loop, which is completely single-stranded, thus demonstrating the in vivo three-strands structure of the human mitochondrial D-loop. Scale bars of 180 nm corresponding to 0.5 kbp are reported on each picture. The position of the D-loop structure is indicated by the asterisk. Source material: U-2 OS cells.

Article Snippet: Using the psoralen-mediated cross-linking conditions indicated in the previous step, around 70–100 units of PvuII-HF enzyme (New England Biolabs, #R3151S) are sufficient to obtain digestion of 10–15 μg of genomic DNA (7–10 units/μg of genomic DNA) in three to five hours at 37°C.

Techniques: In Vivo